Characterization of the mammalian mRNA 3???-end processing complex

描述（由申请人提供）：本提案的长期目标是通过对3‘加工复合物的综合表征，详细了解哺乳动物mRNA 3’加工的机制。mRNA 3′端形成是真核生物基因表达的重要步骤，对RNA代谢的稳定性、输出和翻译等方面有着深远的影响。此外，受调控的mRNA 3'加工已成为基因控制的关键机制。异常的3′加工信号和突变的3′加工因子导致包括癌症在内的许多人类疾病。对于几乎所有的真核mrna， 3'加工包括两个催化步骤，一个核内裂解裂解，然后加入一个聚(a)尾部。这两个步骤都发生在一个大分子机制中，即3'加工复合体。该复合物的结构-功能关系是理解mRNA 3'加工机制的核心问题。我们最近纯化了组装在mRNA靶标上的功能性3'加工复合体，并启动了蛋白质组学、功能和结构分析。以我们的纯化系统为基础，我们将解决该领域的几个基本和长期存在的问题：mrna中的3'加工信号是如何被特异性识别的？3‘加工复合物的蛋白质组成是什么？在3’加工反应中它是如何变化的？3'加工络合物的结构是什么？为了实现我们的目标，我们设计了以下具体目标：在纯化的3'加工复合体内全面绘制蛋白质- rna相互作用图。2. 表征3′加工反应过程中3′加工配合物的组成变化。3. 利用单粒子冷冻电镜（cryo-EM）表征了3′加工配合物在3′加工反应过程中的结构变化。这些研究的完成将为mRNA 3‘加工复体的结构-功能关系和动力学提供新的和重要的机制见解，并可能为开发新的治疗方法提供基础，以治疗由异常3’加工引起的疾病。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing through comprehensive characterization of the 3' processing complex. mRNA 3'-end formation is an essential step of gene expression in eukaryotes, and has profound influences on many aspects of RNA metabolism, including mRNA stability, export, and translation. Additionally, regulated mRNA 3' processing has emerged as a critical mechanism for gene control. Aberrant 3' processing signals and mutant 3' processing factors cause a number of human diseases, including cancer. For almost all eukaryotic mRNAs, 3' processing involves two catalytic steps, an endonucleolytic cleavage followed by the addition of a poly(A) tail. Both of these steps take place in a macromolecular machinery, the 3' processing complex. The structure-function relationship of this complex is a central question in understanding the mechanism of mRNA 3' processing. We have recently purified the functional 3' processing complex assembled on its mRNA target and initiated proteomic, functional, and structural analyses. Building on our purification system, we will address several fundamental and long-standing questions in the field: How are the 3' processing signals in mRNAs specifically recognized? What is the protein composition of the 3' processing complex and (how) does it change during 3' processing reaction? What is the structure of the 3' processing complex? To achieve our objectives, we have designed the following specific aims: 1. Comprehensively map the protein-RNA interactions within the purified 3' processing complex. 2. Characterize the compositional changes of the 3' processing complex during 3' processing reaction. 3. Characterize the structural changes of the 3' processing complex during 3' processing reaction using single particle cryo-electron microscopy (cryo-EM). Accomplishment of the proposed studies will provide new and significant mechanistic insights into the structure-function relationship and the dynamics of the mRNA 3' processing complex, and may also provide the foundation for development of new therapeutic approaches against diseases caused by aberrant 3' processing. PUBLIC HEALTH RELEVANCE: mRNA 3'-end formation is an essential step of eukaryotic gene expression. It plays an important role in gene regulation, and aberrant 3'-end formation has been implicated in a variety of human diseases. The goal of this project is to understand the mechanism of mRNA 3' processing through comprehensive characterization of the 3' processing machinery.